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Robert Huber

Publications and source records attributed to Robert Huber.

At least 145 records · Page 8Linked to original sources
PubMed2002

The structural basis of riboflavin binding to Schizosaccharomyces pombe 6,7-dimethyl-8-ribityllumazine synthase.

Riboflavin is an essential cofactor in all organisms. Its direct biosynthetic precursor, 6,7-dimethyl-8-ribityllumazine, is synthesised by the enzyme 6,7-dimethyl-8-ribityllumazine synthase. Recently, we have found that the enzyme from Schizosaccharomyces pombe binds riboflavin, the final product of the pathway with a relatively high affinity with a KD of 1.2 microM. Here, we report on the crystal structure of lumazine synthase from S. pombe with bound riboflavin and compare the binding mode with those of the substrate analogue inhibitor 5-nitro-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione and of the product analogue 6-carboxyethyl-7-oxo-8-ribityllumazine. In all complexes the pyrimidinedione moieties of each respective ligand bind in a very similar orientation. Binding of riboflavin additionally involves a stacking interaction of the dimethylbenzene moiety with the side-chain of His94, a highly conserved residue in all lumazine synthases. The enzyme from Bacillus subtilis showed a KD of at least 1 mM whereas the very homologous enzyme from Saccharomyces cerevisiae had a comparable KD of 3.9 microM. Structural comparison of the S. cerevisiae, the S. pombe, and the mutant enzymes suggests that fine tuning of affinity is achieved by influencing this stacking interaction.

Amino Acid Sequence↗
PubMed2002

Phosphorylation and flexibility of cyclic-AMP-dependent protein kinase (PKA) using (31)P NMR spectroscopy.

Cell signaling pathways rely on phosphotransfer reactions that are catalyzed by protein kinases. The protein kinases themselves are typically regulated by phosphorylation and concurrent structural rearrangements, both near the catalytic site and elsewhere. Thus, physiological function requires posttranslational modification and deformable structures. A prototypical example is provided by cyclic AMP-dependent protein kinase (PKA). It is activated by phosphorylation, is inhomogeneously phosphorylated when expressed in bacteria, and exhibits a wide range of dynamic properties. Here we use (31)P nuclear magnetic resonance (NMR) spectroscopy to characterize the phosphorylation states and to estimate the flexibility of the phosphorylation sites of 2-, 3-, and 4-fold phosphorylated PKA. The phosphorylation sites Ser10, Ser139, Thr197, and Ser338 are assigned to individual NMR resonances, assisted by complexation with AMP-PNP and dephosphorylation with alkaline phosphatase. Rotational diffusion correlation times estimated from resonance line widths show progressively increasing flexibilities for phosphothreonine 197, phosphoserines 139 and 338, and disorder at phosphoserine 10, consistent with crystal structures of PKA. However, because the apparent rotational diffusion correlation time fitted for phosphothreonine 197 of the activation loop is longer than the overall PKA rotational diffusion time, microsecond to millisecond time scale conformational exchange effects involving motions of phosphothreonine 197 are probable. These may represent "open"-"closed" transitions of the uncomplexed protein in solution. These data represent direct measurements of flexibilities also associated with functional properties, such as ATP binding and membrane association, and illustrate the applicability of (31)P NMR for functional and dynamic characterization of protein kinase phosphorylation sites.

Alkaline Phosphatase↗
PubMed2002

The 1.9-A crystal structure of the noncollagenous (NC1) domain of human placenta collagen IV shows stabilization via a novel type of covalent Met-Lys cross-link.

Triple-helical collagen IV protomers associate through their N- and C-termini forming a three-dimensional network, which provides basement membranes with an anchoring scaffold and mechanical strength. The noncollagenous (NC1) domain of the C-terminal junction between two adjacent collagen IV protomers from human placenta was crystallized and its 1.9-A structure was solved by multiple anomalous diffraction (MAD) phasing. This hexameric NC1 particle is composed of two trimeric caps, which interact through a large planar interface. Each cap is formed by two alpha 1 fragments and one alpha 2 fragment with a similar previously uncharacterized fold, segmentally arranged around an axial tunnel. Each monomer chain folds into two structurally very similar subdomains, which each contain a finger-like hairpin loop that inserts into a six-stranded beta-sheet of the neighboring subdomain of the same or the adjacent chain. Thus each trimer forms a quite regular, but nonclassical, sixfold propeller. The trimer-trimer interaction is further stabilized by a previously uncharacterized type of covalent cross-link between the side chains of a Met and a Lys residue of the alpha 1 and alpha 2 chains from opposite trimers, explaining previous findings of nonreducible cross-links in NC1. This structure provides insights into NC1-related diseases such as Goodpasture and Alport syndromes.

Amino Acid Sequence↗
PubMed2002

Fighting fruit flies: a model system for the study of aggression.

Despite the importance of aggression in the behavioral repertoire of most animals, relatively little is known of its proximate causation and control. To take advantage of modern methods of genetic analysis for studying this complex behavior, we have developed a quantitative framework for studying aggression in common laboratory strains of the fruit fly, Drosophila melanogaster. In the present study we analyze 73 experiments in which socially naive male fruit flies interacted in more than 2,000 individual agonistic interactions. This allows us to (i) generate an ethogram of the behaviors that occur during agonistic interactions; (ii) calculate descriptive statistics for these behaviors; and (iii) identify their temporal patterns by using sequence analysis. Thirty-minute paired trials between flies contained an average of 27 individual agonistic interactions, lasting a mean of 11 seconds and featuring a variety of intensity levels. Only few fights progressed to the highest intensity levels (boxing and tussling). A sequential analysis demonstrated the existence of recurrent patterns in behaviors with some similarity to those seen during courtship. Based on the patterns characterized in the present report, a detailed examination of aggressive behavior by using mutant strains and other techniques of genetic analysis becomes possible.

Aggression↗
PubMed2002

Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine.

Molecular chaperones and proteases monitor the folded state of other proteins. In addition to recognizing non-native conformations, these quality control factors distinguish substrates that can be refolded from those that need to be degraded. To investigate the molecular basis of this process, we have solved the crystal structure of DegP (also known as HtrA), a widely conserved heat shock protein that combines refolding and proteolytic activities. The DegP hexamer is formed by staggered association of trimeric rings. The proteolytic sites are located in a central cavity that is only accessible laterally. The mobile side-walls are constructed by twelve PDZ domains, which mediate the opening and closing of the particle and probably the initial binding of substrate. The inner cavity is lined by several hydrophobic patches that may act as docking sites for unfolded polypeptides. In the chaperone conformation, the protease domain of DegP exists in an inactive state, in which substrate binding in addition to catalysis is abolished.

Crystallography, X-Ray↗
PubMed2002

New isolates and physiological properties of the Aquificales and description of Thermocrinis albus sp. nov.

The ecology of the Aquificales was studied using a combination of phylogenetic and cultivation approaches. Enrichment cultures were prepared from low-salt and marine samples of geothermally and volcanically heated environments of the United States (Yellowstone National Park), Russia (Kamchatka), Italy, Germany, Djibouti, Iceland, and Africa (Lake Tanganyika). Isolation of single cells using the selected cell cultivation technique resulted in 15 different pure cultures. Comparisons of their 16S rRNA gene sequences showed that most of the isolates were new representatives of the major lineages of the Aquificaceae, represented by the genera Aquifex, Thermocrinis, Hydrogenobaculum, and Hydrogenobacter. Isolate HI 11/12, which was obtained from whitish streamers in the Hveragerthi area of Iceland, represents a separate branch within the Aquificaceae. The organism grew at salinities up to 0.7% NaCl and at temperatures up to 89 degrees C. Depending on the culture conditions, the organisms occurred as single motile rods, as aggregates, or as long filaments that formed whitish streamer-like cell masses. The novel isolate grew chemolithoautotrophically with hydrogen, sulfur, or thiosulfate as the electron donor under microaerophilic conditions. It represents a second species within the order Thermocrinis, which we name Thermocrinis albus HI 11/12 (DSM 14484, JCM 11386).

Bacteria↗
PubMed2002

The methyl group of N(alpha)(Me)Arg-containing peptides disturbs the active-site geometry of thrombin, impairing efficient cleavage.

Bivalent peptidic thrombin inhibitors consisting of an N-terminal d-cyclohexylalanine-Pro-N(alpha)(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with K(i) values in the picomolar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased stability, the most potent of these inhibitors, I-11 (K(i)=37pM), containing an N(alpha)(Me)Arg-Thr bond, was crystallized in complex with human alpha-thrombin. X-ray data were collected to 1.8A resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N(alpha)(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 O(gamma) atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 O(gamma) are separated from each other, impairing the nucleophilic attack of the Ser195 O(gamma) toward the N(alpha)(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate.

Arginine↗
PubMed2002

Characterization of the residues involved in the human alpha-thrombin-haemadin complex: an exosite II-binding inhibitor.

Haemadin is a 57-amino acid thrombin inhibitor from the land-living leech Haemadipsa sylvestris, whose structure has recently been determined in complex with human alpha-thrombin. Here we communicate the effect of ionic strength on the kinetics of the inhibition of human alpha-thrombin by haemadin, by using thrombin mutants modified in exosite II. Data analysis has allowed both the ionic and nonionic binding contributions to be ascertained, with the nonionic component being virtually the same for all of the thrombins that have been examined, while the ionic binding energy contributions varied from molecule to molecule. In the case of the native human alpha-thrombin-haemadin complex, ionic interactions contribute -17 kJ/mol to the Gibbs free energy of binding, this being the equivalent of up to six salt bridges. These salt bridges make up 20% of the total binding energy at zero ionic strength, and this has been attributed to the C-terminal tail alone. In addition, the contributions of the N-terminal and C-terminal regions of haemadin to its tight binding have been ascertained by using derivatives of both haemadin and thrombin. Limited proteolysis using formic acid produced haemadin cleaved between residues 40 and 41, removing the majority of the C-terminal tail. This truncated haemadin displayed a 20000-fold reduced affinity for thrombin, and was no longer a tight binding inhibitor. A form of thrombin in which the active site serine has been blocked by diisopropyl fluorophosphate binds to haemadin, but with a 72000-fold reduced affinity, indicating that the N-terminus is more important than the C-terminus for strong binding.

Amino Acid Sequence↗
PubMed2002

Reaction mechanism of GTP cyclohydrolase I: single turnover experiments using a kinetically competent reaction intermediate.

GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopterin 3'-triphosphate, which is the first committed precursor of tetrahydrofolate and tetrahydrobiopterin. The kinetically competent reaction intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone, was used as substrate for single turnover experiments monitored by multiwavelength photometry. The early reaction phase is characterized by the rapid appearance of an optical transient with an absorption maximum centred at 320. This species is likely to represent a Schiff base intermediate at the initial stage of the Amadori rearrangement of the carbohydrate side-chain. Deconvolution of the optical spectra suggested four linearly independent processes. A fifth reaction step was attributed to photodecomposition of the enzyme product. Pre-steady state experiments were also performed with the H179A mutant which can catalyse a reversible conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone but is unable to form the final product, dihydroneopterin triphosphate. Optical spectroscopy failed to detect any intermediate in the reversible reaction sequence catalysed by the mutant protein. The data obtained with the wild-type and mutant protein in conjunction with earlier quenched flow studies show that the enzyme-catalysed opening of the imidazole ring of GTP and the hydrolytic release of formate from the resulting formamide type intermediate are both rapid reactions by comparison with the subsequent rearrangement of the carbohydrate side-chain which precedes the formation of the dihydropyrazine ring of dihydroneopterin triphosphate.

Catalysis↗
PubMed2002

Crystal structure of the Bse634I restriction endonuclease: comparison of two enzymes recognizing the same DNA sequence.

Crystal structures of Type II restriction endonucleases demonstrate a conserved common core and active site residues but diverse structural elements involved in DNA sequence discrimination. Comparative structural analysis of restriction enzymes recognizing the same nucleotide sequence might therefore contribute to our understanding of the structural diversity of specificity determinants within restriction enzymes. We have solved the crystal structure of the Bacillus stearothermophilus restriction endonuclease Bse634I by the multiple isomorphous replacement technique to 2.17 A resolution. Bse634I is an isoschisomer of the Cfr10I restriction enzyme whose crystal structure has been reported previously. Comparative structural analysis of the first pair of isoschisomeric enzymes revealed conserved structural determinants of sequence recognition and catalysis. However, conformations of the N-terminal subdomains differed between Bse634I/Cfr10I, suggesting a rigid body movement that might couple DNA recognition and catalysis. Structural similarities extend to the quaternary structure level: crystal contacts suggest that Bse634I similarly to Cfr10I is arranged as a tetramer. Kinetic analysis reveals that Bse634I is able to interact simultaneously with two recognition sites supporting the tetrameric architecture of the protein. Thus, restriction enzymes Bse634I, Cfr10I and NgoMIV, recognizing overlapping nucleotide sequences, exhibit a conserved tetrameric architecture that is of functional importance.

Base Sequence↗
PubMed2002

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Aquifex aeolicus.

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Homologues of complex I are present in the three domains of life. Here, we report the properties of complex I in membranes of the hyperthermophilic bacterium Aquifex aeolicus. The complex reacted with NADH but not with NADPH and F(420)H(2) as electron donors. Short-chain analogues of ubiquinone like decyl-ubiquinone and ubiquinone-2 were suitable electron acceptors. The affinities towards NADH and ubiquinone-2 were comparable to the ones obtained with the Escherichia coli complex I. The reaction was inhibited by piericidin A at the same concentration as in E. coli. The complex showed an unusual pH optimum at pH 9 and a maximal rate at 80 degrees C. We found no evidence for the presence of an alternative, single subunit NADH dehydrogenase in A. aeolicus membranes. The NADH:ferricyanide reductase activity of detergent extracts of A. aeolicus membranes sedimented as a protein with a molecular mass of approximately 550 kDa. From the data we concluded that A. aeolicus contains a NADH:ubiquinone oxidoreductase resembling complex I of mesophilic bacteria.

Amino Acid Sequence↗
PubMed2002

Chronic alterations in serotonin function: dynamic neurochemical properties in agonistic behavior of the crayfish, Orconectes rusticus.

The biogenic amine serotonin [5-hydroxytryptamine (5-HT)] has received considerable attention for its role in behavioral phenomena throughout a broad range of invertebrate and vertebrate taxa. Acute 5-HT infusion decreases the likelihood of crayfish to retreat from dominant opponents. The present study reports the biochemical and behavioral effects resulting from chronic treatment with 5-HT-modifying compounds delivered for up to 5 weeks via silastic tube implants. High performance liquid chromatography with electrochemical detection (HPLC-ED) confirmed that 5,7-dihydroxytryptamine (5,7-DHT) effectively reduced 5-HT in all central nervous system (CNS) areas, except brain, while a concurrent accumulation of the compound was observed in all tissues analyzed. Unexpectedly, two different rates of chronic 5-HT treatment did not increase levels of the amine in the CNS. Behaviorally, 5,7-DHT treated crayfish exhibited no significant differences in measures of aggression. Although treatment with 5-HT did not elevate 5-HT content in the CNS, infusion at a slow rate caused animals to escalate more quickly while 5-HT treatment at a faster rate resulted in slower escalation. 5,7-DHT is commonly used in behavioral pharmacology and the present findings suggest its biochemical properties should be more thoroughly examined. Moreover, the apparent presence of powerful compensatory mechanisms indicates our need to adopt an increasingly dynamic view of the serotonergic bases of behavior like crayfish aggression.

5,7-Dihydroxytryptamine↗
PubMed2002

The 1.4 a crystal structure of kumamolysin: a thermostable serine-carboxyl-type proteinase.

Kumamolysin is a thermostable endopeptidase from Bacillus novosp. MN-32, exhibiting maximal proteolytic activity around pH 3. It belongs to the newly identified family of serine-carboxyl proteinases, which also includes CLN2, a human lysosomal homolog recently implicated in a fatal neurodegenerative disease. Kumamolysin and its complexes with two aldehyde inhibitors were crystallized, and their three-dimensional structures were solved and refined with X-ray data to 1.4 A resolution. As its Pseudomonas homolog, kumamolysin exhibits a Ser/Glu/Asp catalytic triad with particularly short interconnecting hydrogen bonds and an oxyanion hole enabling the reactive serine to attack substrate peptide bonds at quite acidic pH. An additional Glu/Trp pair, unique to kumamolysin, might further facilitate proton delocalization during nucleophilic attack, in particular at high temperature.

Amino Acid Sequence↗
PubMed2002

Crystal structure of the dinuclear zinc aminopeptidase PepV from Lactobacillus delbrueckii unravels its preference for dipeptides.

PepV from Lactobacillus delbrueckii, a dinuclear zinc peptidase, has been characterized as an unspecific amino dipeptidase. The crystal structure of PepV in complex with the phosphinic inhibitor AspPsi[PO(2)CH(2)]AlaOH, a dipeptide substrate mimetic, reveals a "catalytic domain" and a "lid domain," which together form an internal active site cavity that traps the inhibitor. The catalytic domain is topologically similar to catalytic domains from amino- and carboxypeptidases. However, the lid domain is unique among the related enzymes. In contrast to the other related exopeptidases, PepV recognizes and fixes the dipeptide backbone, while the side chains are not specifically probed and can vary, rendering it a nonspecific dipeptidase. The cocrystallized inhibitor illustrates the two roles of the two catalytic zinc ions, namely stabilization of the tetrahedral intermediate and activation of the catalytic water molecule.

Amino Acid Sequence↗
PubMed2002

Gene sequence and the 1.8 A crystal structure of the tungsten-containing formate dehydrogenase from Desulfovibrio gigas.

Desulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H(+) by buried waters and protonable amino acids and for CO(2) through a hydrophobic channel.

Amino Acid Sequence↗
PubMed2002

Studies on the reaction mechanism of riboflavin synthase: X-ray crystal structure of a complex with 6-carboxyethyl-7-oxo-8-ribityllumazine.

Riboflavin synthase catalyzes the disproportionation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. We have determined the structure of riboflavin synthase from Schizosaccharomyces pombe in complex with the substrate analog, 6-carboxyethyl-7-oxo-8-ribityllumazine at 2.1 A resolution. In contrast to the homotrimeric solution state of native riboflavin synthase, we found the enzyme to be monomeric in the crystal structure. Structural comparison of the riboflavin synthases of S. pombe and Escherichia coli suggests oligomer contact sites and delineates the catalytic site for dimerization of the substrate and subsequent fragmentation of the pentacyclic intermediate. The pentacyclic substrate dimer was modeled into the proposed active site, and its stereochemical features were determined. The model suggests that the substrate molecule at the C-terminal domain donates a four-carbon unit to the substrate molecule bound at the N-terminal domain of an adjacent subunit in the oligomer.

Amino Acid Sequence↗
PubMed2002

Application of NMR in structural proteomics: screening for proteins amenable to structural analysis.

In the time of structural proteomics when protein structures are targeted on a genome-wide scale, the detection of "well-behaved" proteins that would yield good quality NMR spectra or X-ray images is the key to high-throughput structure determination. Already, simple one-dimensional proton NMR spectra provide enough information for assessing the folding properties of proteins. Heteronuclear two-dimensional spectra are routinely used for screenings that reveal structural, as well as binding, properties of proteins. NMR can thus provide important information for optimizing conditions for protein constructs that are amenable to structural studies.

Nuclear Magnetic Resonance, Biomolecular↗